Comprehensive Essentiality Analysis of the Genome by Saturation Transposon Mutagenesis and Deep Sequencing

() is an opportunistic pathogen that is frequently isolated from urban water systems, posing a health risk to susceptible individuals. Despite its ability to cause tuberculosis-like pulmonary disease, very few studies have probed the genetics of this opportunistic pathogen. Here, we report a comprehensive essentiality analysis of the genome. Deep sequencing of a high-density library of transposon mutants revealed that 86.8% of the chromosomal thymine-adenine (TA) dinucleotide target sites were permissive to insertion, leaving 13.2% TA sites unoccupied. Our analysis identified 394 of the 5,350 annotated open reading frames (ORFs) as essential. The majority of these essential ORFs (84.8%) share essential mutual orthologs with (). A comparative genomics analysis identified 139 essential ORFs that share essential orthologs in four other species of mycobacteria. Thirteen essential ORFs share orthologs in all four species that were identified as being not essential, while only two essential ORFs are absent in all species compared. We used the essentiality data and a comparative genomics analysis reported here to highlight differences in essentiality between candidate drug targets and the corresponding orthologs. Our findings suggest that the genome encodes redundant or additional pathways that may confound validation of potential drugs and drug target candidates against the opportunistic pathogen. Additionally, we identified 57 intergenic regions containing four or more consecutive unoccupied TA sites. A disproportionally large number of these regions were located upstream of / genes. Finally, we present an essentiality and orthology analysis of the pRAW-like plasmid, pMK1248. IMPORTANCE is one of the most common nontuberculous mycobacterial pathogens associated with tuberculosis-like pulmonary disease. Drug resistance emergence is a threat to the control of infections, which already requires long-term, multidrug courses. A comprehensive understanding of biology is critical to facilitate the development of new and more efficacious therapeutics against . We combined transposon-based mutagenesis with analysis of insertion site identification data to uncover genes and other genomic regions required for growth. We also compared the gene essentiality data set of to those available for several other mycobacteria. This analysis highlighted key similarities and differences in the biology of compared to these other species. Altogether, the genome-wide essentiality information generated and the results of the cross-species comparative genomics analysis represent valuable resources to assist the process of identifying and prioritizing potential drug target candidates and to guide future studies on biology.